Association of interleukin-17F (rs763780) single nucleotide polymorphism with multiple sclerosis and optic neuritis

IL-17F single nucleotide polymorphism (SNP) can affect IL-17F expression and activity and this can lead to the increased susceptibility to several autoimmune diseases. The aim was to investigate the association of IL-17F (rs763780) SNP with the development of multiple sclerosis (MS) in a cohort of Egyptian patients and to evaluate the effect of this polymorphism on the disease course. IL-17F (rs763780) gene polymorphisms was typed by TaqMan genotyping assay for 231 Egyptians divided into 102 MS patients and 129 healthy controls with matched age and sex. The IL-17F rs763780 C containing genotypes (CT+CC) and C allele have statistically significant increased frequency in MS patients when compared with controls (p = 0.005 and 0.004 respectively) especially in females’ patients (p = 0.005 and 0.006 respectively). The heterozygous CT genotype was associated with the presence of optic neuritis (p = 0.038). The multivariable regression analysis revealed significant associations between smoking, the higher frequency of attacks and the prediction of higher EDSS score (p = 0.032, 0.049 respectively). It can be concluded that the IL-17F rs763780 C containing genotypes (CT and CC) and C allele may be risk factors for the development of MS in the studied Egyptian cohort by a gender-dependent mechanism that contributes to tendency for predisposition in females and optic neuritis is more common in patients carrying the CT heterozygous genotype.


Materials and methods
This is a case control study that was conducted on 231 individuals (102 MS patients and 129 apparently healthy control subjects) matched in age and sex.The patients age ranged from 18 to 54 years with (mean ± SD = 32.3 ± 8 y) with 41 males and 61 females.The control age ranged from 17 to 54 years with (mean ± SD = 31.3± 4.7 y), with 53 males and 76 females.All patients gave informed consent to their participation in this study, and the research was approved by the institutional research board (IRB) under number (MD/169).
MS was diagnosed according to McDonald 2017 criteria for Diagnosis of MS 18 .The patient was subjected to full history and complete neurological examination with special emphasis on the presence of multiple sclerosisassociated symptoms (mobility problems, vision problems, problems with coordination, cognitive dysfunction, fatigue, and pain), as well as duration of the disease.All patients were subjected to Magnetic resonance imaging (MRI) to confirm the diagnosis of MS.
The MS disease course was classified into four major categories 1 .Relapsing-remitting MS (RRMS), it is marked by flare-ups (relapses or exacerbations) of symptoms followed by periods of remission when symptoms improve or disappear.Secondary progressive MS (SPMS), SPMS follows an initial RRMS and characterized by progressive worsening of neurologic function (accumulation of disability) over time.Primary progressive MS (PPMS) is characterized by worsening neurologic function (accumulation of disability) from the onset of symptoms with no relapses or remissions.Progressive-relapsing MS (PRMS) is progressive from the start, with intermittent flare-ups of worsening symptoms along the way and with no periods of remission.
Determination of expanded disability status scale (EDSS) score was done to measure the severity of MS.The EDSS score ranges from 0 to 10 in 0.5-unit increments.A score of 0 means no signs or symptoms; score of ≤ 3 represent mild disability with no or minimal impairment of ambulation, score of 3 to 6 refer to moderate to relatively severe disability and impairment of gait and daily routine but patient still able to walk; score of ≥ 6 refers to more severe disability and requiring assistance with walking, while an EDSS of 10 refers to death related to MS 19 .Exclusion criteria of cases were history of chronic CNS diseases, other autoimmune diseases, Children and refused patients.
Two and half ml of venous blood were collected from all controls and patients by clean venipuncture using plastic disposable syringes then delivered into 5 ml sterile vacutainer tubes containing 50 µl tri-potassium EDTA solutions and stored at − 20 °C until DNA extraction was done.

Genotyping of IL-17F rs763780
The studied SNP comprised of C and T alleles.It is located on chromosome 6 on IL17F gene.Allele change from CAT into CGT leads to amino acid change from histidine into arginine.Table 1 Genomic DNA was extracted from whole venous EDTA blood using Thermo Scientific Gene JET whole Blood Genomic DNA Purification Mini Kits.DNA concentration was measured by NanoDrop 2000c (thermo Scientific, USA).Then the extracted DNA samples were stored at − 20 °C until use.

TaqMan SNP genotyping assays (real time PCR)
Genotyping was done by real-time PCR using TaqMan SNP genotyping assay technique (Applied Biosystem, lot 4440042, Foster, USA).The SNP ID is C_2234166_10 for IL-17F rs763780 and the chromosomal location is Chr.6: 52101739.One probe labeled with VIC ® dye detects the Allele C sequence & the other probe labeled with FAM™ dye detects the Allele T sequence.The context Sequence [VIC/FAM] was GTG GAT ATG CAC CTC TTA CTG CAC A[C/T] GGT GGA TGA CAG GGG TGA CGC AGG T. Twenty microliter of PCR reaction mix for qPCR analysis was prepared as follow: 10.0 ul TaqMan ® universal master mix II with UNG, 2X (Applied Biosystem, lot 4440042), 1.0 ul TaqMan ® assay 20X and 5.0 µl DNA template + 4 ul RNase-free water.For each sample, twenty ul of PCR reaction mix was transferred to the 48-well Reaction plate.The plate was sealed using appropriate cover and was briefly centrifuged and loaded into the real-time PCR (Step one, Applied Biosystem, USA).Reaction conditions were carried out with the following cycling stages; stage I for UNG incubation at 60 °C for 30 s; stage II for polymerase activation at 95 °C for 10 min; stage III for PCR and includes 50 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 90 s and finally stage IV 60 °C for 30 s. Allelic discrimination was carried out by measuring fluorescence intensity at the endpoint.The SDS software version 1.7 (Applied Biosystem, Foster, USA) was used for typing of genotypes and alleles of the SNPs.Each sample was interpreted according to the increase in fluorescence intensity of either FAM (TT homozygous genotype) or VIC (CC homozygous genotype) or both FAM and VIC (CT heterozygous genotype).Ten percent of samples were repeated for confirmation.

Statistical analysis
The statistical analysis of data was done using excel program (Microsoft Office 2013) and IBM SPSS (statistical package for social science) program (SPSS, Inc, Chicago, IL) version 20.Qualitative data were presented as frequency and percentage.Chi square and Fisher's exact tests were used to compare groups.Quantitative data were presented by mean, ± SD, median and range.Comparisons between two groups were done using t-test or Man Whitney (for non-parametric).Deviations from Hardy-Weinberg equilibrium expectations were determined using the chi-square test Goodness-of-fit test.Both cases and control groups were found to be in agreement with Hardy Weinberg equilibrium.Linear regression analysis was computed using generalized linear model.Odds ratio and 95% confidence interval were calculated.N.B: p is significant if < 0.05 at confidence interval 95%.

Ethical approval and consent to participate
The study was approved by the local Ethical Committee: Institutional Research Board (IRB) of Mansoura Faculty of Medicine (MD/169) and All methods were performed in accordance with the ethical standards as laid down in the Declaration of Helsinki and its later amendments or comparable ethical standards.Written informed consent was obtained from all participants from a parent and/or legal guardian.

Results
The clinical characteristics of MS patients are presented in Table 2.The distribution of IL-17F rs763780 genotype and allele frequencies showed significantly higher frequency of TT genotype (69% vs. 51%, p = 0.005), and T allele (83.7% vs. 72.5%,p = 0.004) among control group.In MS patients, a significant increased frequency was observed for the CT (43.1% vs. 29.5%,p = 0.015), CC (5.9% vs. 1.6%, p = 0.045), CT+CC (49% vs. 31.1%,p = 0.005) genotypes and for the C allele (27.5% vs. 16.3%, p = 0.004) Table 3.At the same time, the distribution of the genotypes and alleles of IL-17F rs763780 was analyzed in patients and controls based on gender.In comparison with controls, the female patients had statistically significant higher frequency of CT, CT+CC genotypes and C allele (p = 0.008, 0.005 and 0.006 respectively).Table 4 The distribution of IL-17F rs763780 genotypes was compared in relation to the clinical data of MS patients and non-statistically significant differences were found (p > 0.05) except for the significant association between the heterozygous CT genotype and presence of optic neuritis (p = 0.038).Table 5 Regression analysis for the prediction of higher EDSS score was done using age, gender, smoking, age of disease onset, duration of disease, total number of attacks and IL-17F (rs763780) genotypes as covariates and presented in Table 6.The multivariable analysis revealed significant associations between smoking, and higher frequency of attacks and the prediction of higher EDSS score (p = 0.032, 0.049 respectively).However, in multivariable analysis, smoking as well as higher frequency of attacks are considered as bad predictors of higher EDSS score (p = 0.032, 0.049 respectively).

Discussion
MS is one of the autoimmune chronic neuro-inflammatory demyelinating diseases of the CNS that is a common cause of serious physical disability in young adults, especially women 20 .The MS brain and nerve damage is due to CNS inflammation.Although, the exact etio-pathogenic factor that initiates the inflammation is unknown, however, infectious, genetic, and environmental factors have been suggested 21 .Hashem et al. 22 meta-analysis reported the prevalence of MS to be 14.1/100,000 in Egyptian and 25/100.000by El-Sawy et al. 23 .While T helper 1 (Th1) proinflammatory cytokines, IFN-γ, TNF-α, IL-1, IL-2, and IL-12, have been linked to the onset and perpetuation of MS, the Th2 anti-inflammatory cytokines, IL-4, IL-10 and TGF-β, have been linked to MS remission 24 .It was known that the MS disease is a Th1-cell-driven.Earlier, Th17 cells have been considered as important players in the immune pathogenesis of MS 25 .IL-17 transcription is up-regulated in the MS lesion 26 , IL-17F levels are elevated in the serum and CSF from patients with active MS 27 and memory T cells producing IL-17 infiltrate into MS lesions 28 .IL-17F, a member of IL-17 family, is a pro-inflammatory cytokine and induces the expression of various cytokines, chemokines, and adhesion molecules.Therefore, it is proved to have a crucial role in the emergence of autoimmune diseases 29 .
Liu et al. 30 analyzed the association between IL-17 genetic variants and susceptibility to human diseases.They identified significant associations between 18 human diseases and seven genetic variants in IL-17.The strongest evidence was between IL-17A rs2275913, IL-17A rs8193037, IL-17F rs1889570, IL-17F rs763780 and susceptibility to lung and cervical cancer, spondylarthritis, asthma, MS, RA.The bioinformatics analysis suggested that these variants may be associated with or fall in putative functional regions.The explanation for such association may be due to increased expression of proinflammatory cytokines, chemokines, and growth factors.Additionally, IL-17F rs763780 SNP could trigger high IL-17 expression which increases the risk of the abovementioned human diseases.www.nature.com/scientificreports/ In the present study, the IL-17F rs763780 CT, CC genotypes and C allele were significantly higher in MS patients especially the females and TT genotype and T allele were higher in the healthy controls.These data suggest that the C allele and C containing genotypes may be susceptibility risk factors for the development of MS and the T allele and TT genotype may be protective factors.At the same time, the heterozygous CT genotype was associated with presence of optic neuritis and smoking, longer duration of disease and higher frequency of attacks can be considered as predictor of higher EDSS score.
Atya et al. 31 and El Sharkawi et al. 32 work on Egyptian reported a significant increased frequency of IL-17F rs763780 CT+CC genotypes and C allele in MS patients and between CT genotype and the delay in disease onset.In contrast, non-significant associations were found between the studied SNP and progression index (PI), or total number of relapses.They also reported an increased frequency of the CT genotype in female patients.This observation was concordant with reported predominance of most autoimmune diseases in female's especially autoimmune disorders of CNS.This can be attributed to hormonal effects, genetic factors, and exposure to different environmental factors which may explain the differences between male and female as regard the immune system, CNS, and response to immunotherapy 33 .
On contrary, Wang et al. 16 reported a significantly increased frequency of rs763780 TT genotype and T allele in Chinese Han patients MS patients.The difference between the results of the present study and those of Chinese Han population can be explained by; different sample size; different genotyping method (Real time-PCR vs. RFLP); different environmental exposure; different ethnic background.Manni et al. 34 in their study on the Y-chromosome gene pool in the modern Egyptian population confirmed the mixture of European, Middle Eastern and African genetic characteristics in Egyptians genetics.More specifically, analysis of Y-chromosome haplotypes demonstrated different distribution of different haplotype between Upper Egypt and Lower Egypt 35 , i.e., different ethnic origins of both populations 36 .
The present study showed a significant association between the heterozygous CT genotype and optic neuritis.The progressive course of MS may lead to more inflammation and damage of the optic nerve.Henderson et al. 37 used optical coherence tomography (OCT), giving an idea of axonal loss by measuring of the retinal nerve fiber layer (RNFL) and macular volume.They found a significant decrease in the mean of RNFL thickness and macular volume in SPMS group when compared with controls.Tian et al. 38 investigated IL-17 level and gene expression in the optic nerve to explore the role of Th17 in the pathogenesis of optic neuritis in EAE in mice.They found the concentrations of IL-17 protein in the optic nerve were significantly up-regulated at 11 days post-immunization and mRNA expression of IL-17 was consistent with their protein expression.They indicated that IL-17 may mediate inflammatory pathogenicity at the early stage of optic neuritis.Tian et al. 38 findings could explain the association between IL-17F rs763780 and optic neuritis in the studied Egyptian MS patients.The increased expression and serum level of IL-17 in optic nerve may be involved in the immune-inflammatory pathogenesis of optic neuritis.
Univariable and multivariable regression analysis showed higher scores of EDSS in MS smoker's patients and this was concordant with findings in many epidemiological studies.Hernan et al. 39 reported that smoking increases the risk of conversion from RRMS course to SPMS course.They estimated that the risk to be higher three times or more in smokers.Also, Sundstrom and Nystrom 40 estimated the effect of smoking on the risk of MS progression on newly diagnosed MS cases.They found that smokers were more likely to have progressive disease, and early onset of progression was higher with patients starting smoking before age 15 years.Therefore, cigarette smoking may transform, or accelerate the transformation of the disease into progressive forms.The association can be explained by the increased free radicles and nitric oxide in CNS 41 and chronic cyanide intoxication leading to widespread demyelination 39 .

Conclusion
It can be concluded that the IL-17F rs763780 C containing genotypes (CT and CC) and C allele may be risk factors for the development of MS in the studied Egyptian cohort by a gender-dependent mechanism that contributes to tendency for predisposition in females and optic neuritis is more common in patients carrying the CT heterozygous genotype.

Table 1 .
Genetic characteristics of studied polymorphism.

Table 2 .
Clinical characteristics of MS patients.

Table 3 .
Distribution of IL-17F rs763780 genotypes and alleles in MS patients and healthy controls.OR odds ratio, CI confidence intervals, HWE Hardy Weinberg equation.*Significant (P value < 0.05).

Table 5 .
Relation between genotype frequencies of IL-17F rs763780 and clinical data of MS patients.P1, comparison between TT, TC and CC; p2, comparison between CT+CC versus TT.

Table 6 .
Regression analysis for prediction of EDSS scores.OR odds ratio, CI confidence interval; ordinal regression was used.*Significant (P value < 0.05).